The prolonged lifespan of neurons allows them to participate in the vast information networks underlying all nervous abilities. To ensure longevity and maintain integrity neurons are certain to be endowed with elaborate housekeeping systems, but these maintenance and repair functions are only poorly understood. We wish to understand one of the major maintenance tasks in the nervous system: the prevention of the buildup of spontaneously damaged protein. Without preventive measures such a buildup would be inevitable given the chemical susceptibility of proteins to damage under the temperature and chemical conditions of neuronal tissue. This problem is complex and intellectually rich because the neuron evidently can repair or destroy its damaged proteins without disrupting the integrity of its many molecular assemblies. This problem is also highly relevant to human health because symptoms of impaired and aberrant protein degradation are present in neurological disease, specifically Alzheimer's dementia. Our proposal is to determine the molecular events that govern the fate of neuronal protein molecules. These molecular events will be established in neuronally differentiated cells of the well-described PC12 line, allowing questions on protein fate to be addressed in large populations of clonal and therefore biochemically identical neuronal cells. First, we will determine the lifespan of cell proteins to determine which proteins are turned over rapidly, and at the other extreme which proteins persist for the lifetime of the cell. We will also test whether periods of intense neurosecretory activity alter the lifespan of proteins involved in secretion since active use of a protein may accelerate its damage. Second, we will identify the proteins in neuronally differentiated PC12 cells that are methylated by an enzyme, protein D-aspartyl/L-isoaspartyl methyltransferase (PDM), the only neuronal enzyme known to exclusively modify damaged proteins. Identification of these methylated proteins will show which types of damaged proteins have their fate affected by the methylation pathway and will indicate which functional systems in neurons are especially dependent on methylation for repair or degradation of damaged proteins. Third, we will measure the dynamics of protein methylation and demethylation to establish the number of times that a protein is methylated during its lifetime. The effect of neurosecretion on methylation and demethylation will also be measured to test whether intense secretory activity may be a stress that is followed by a period of repair and degradation initiated by protein methylation. Fourth, we will test the fate of damaged neuronal proteins in cells which are impaired in methylation activity since it can be predicted that blocking the PDM activity of neuronal cells will lead to hyperaccumulation of damaged protein.